An immunoglobulin (antibody) is a physiologically active substance responsible for an immune response. Recently, availability of immunoglobulin has been increased in applications such as medicines, diagnostic agents and separation/purification materials for the corresponding antigen protein. An antibody is taken from the blood of an immunized animal, a culture solution of a cell having antibody producibility or an ascitic fluid culture solution of the animal. However, such blood and a culture solution containing the antibody contain proteins other than the antibody or intricate contaminants derived from a raw-material solution used in the cell culture. Thus, to separate and purify the antibody from these impurity components, a complicated and time-consuming operation is usually required.
Liquid chromatography is important for separating and purifying an antibody. Examples of a chromatographic method for separating an antibody include gel filtration chromatography, affinity-chromatography, ion exchange chromatography and reverse phase chromatography. An antibody is separated and purified by a combination of these methods.
The ion exchange chromatography is a method of separating a counter ion present in a mobile phase by reversibly adsorbing it by an ion exchange group, which is present on the surface of an adsorbent and serves as a stationary phase. As the shape of the adsorbent, beads, flat film and a film such as a hollow fiber, are employed. These base materials, to which a cation exchange group or an anion exchange group is bound, are commercially available as adsorbents. The adsorbent having a cation exchange group, which has a property of mainly adsorbing an antibody and not adsorbing most of other contaminants, has a property of easily concentrating and separating an antibody.
Cation exchange groups are roughly divided into a weak cation exchange group such as a carboxyl group and a strong cation exchange group such as a sulfonic acid group. An adsorbent having a weak cation exchange group has a drawback in that the surface charge of the adsorbent changes as the pH of a mobile phase changes, with the result that a binding capacity to an antibody changes. Accordingly, if an adsorbent having a weak cation exchange group is used for separation/purification of an antibody, the reproducibility of separation becomes poor and the recovery rate of the antibody may decrease. In contrast, in an adsorbent having a strong cation exchange group, since the surface charge of the adsorbent does not change even if the pH of a mobile phase changes, the binding capacity to an antibody does not easily change. In industrial antibody separation/purification processes, although it is difficult to keep the pH of a mobile phase at a constant value, reproducibility of separation is stringently required. For this reason, an adsorbent having a strong cation exchange group is used.
In conventional adsorbents having an ion exchange group, a physiologically active substance adsorbed is generally eluted by increasing the salt concentration of a mobile phase. However, it is known that a physiologically active substance serving as a component of a biomedicine and the like may cause an irreversible change (denaturation) by changing a salt concentration (ion strength) of a mobile phase. Extreme care must be taken to determine these elution conditions. In addition, physiologically active substances are mostly separated and purified in sites (low-temperature chambers) controlled at low temperatures; however, when a physiologically active substance adsorbed is eluted in a mobile phase of a high salt-concentration, there is a risk that a salt precipitated at a low temperature causes clogging of a pipe and a column.
Then, to solve a problem of conventional adsorbents having an ion exchange group, a temperature responsive adsorbent is proposed, from which a physiologically active substance adsorbed can be eluted not by increasing the salt concentration of a mobile phase but by changing an efficient surface density of ion exchange group by temperature.
Patent Literature 1 discloses a packing material containing a charged copolymer, a method for producing the same and a temperature responsive chromatography using the same, in which an efficient surface charge density of a stationary phase can foe changed by temperature change. Patent Literature 2 discloses a temperature responsive chromatographic carrier prepared by densely immobilizing a polymer capable of changing hydration force within a temperature range of 0 to 80° C. to a base material surface by an atom transfer radical polymerization method. Patent Literature 3 discloses a method for producing a temperature responsive chromatographic carrier comprising growing a charged polymer capable of changing hydration force within a temperature range of 0 to 80° C. in accordance with a reaction of an atom transfer radical method using isopropyl alcohol as a solvent. Patent Literature 4 discloses a method for producing a liquid chromatographic carrier, which is prepared by covering a solid surface with a charged polymer capable of changing hydration force within a temperature range of 0 to 80° C., and which is capable of separating a high-molecular weight physiologically active substance useful in the fields of e.g., biology, medicine and pharmacy under specific conditions including an aqueous mobile phase. Non Patent Literature 1 discloses a temperature responsive chromatographic carrier having a carboxyl group and prepared by an atom transfer radical polymerization method and a process thereof. In the Literature, monomer compositions for use in an atom transfer radical polymerization method are disclosed including a monomer composition optimized for lysozyme separation.